This invention relates to the field of polynucleotide sequence determination, in particular, relates to determine the identity of a single nucleotide in a target polynucleotide sequence, e.g., single nucleotide polymorphism (xe2x80x9cSNPxe2x80x9d) analysis.
Techniques for the analysis of polynucleotide sequences have found widespread use in basic research, diagnostics, and forensics. Single nucleotide detection is applied in processes including the detection of single nucleotide polymorphisms, identification of single base changes, speciation, determination of viral load, genotyping, medical marker diagnostics, and the like.
Single nucleotide detection can be accomplished by a number of methods. Most methods rely on the use of the polymerase chain reaction (PCR) to amplify the amount of target DNA. One of the first developed PCR-dependent methods is restriction site polymorphism detection, where the PCR product is cleaved by a restriction enzyme and then analyzed by electrophoresis. Another early method is allele-specific PCR in which one of the PCR primers is designed such that it will discriminate at its 3xe2x80x2 end between DNA targets having a sequence that perfectly matches the primer from those targets not perfectly matching the primer.
TaqMan was the first homogenous assay capable of detecting single nucleotide polymorphisms (U.S. Pat. No. 5,723,591). In this assay, two PCR primers flank a central probe oligonucleotide. The probe oligonucleotide comprises two fluorescent moieties. During the polymerization step of the PCR process, the polymerase cleaves the probe oligonucleotide. The cleavage causes the two fluorescent moieties to become physically separated, which causes a change in the wavelength of the fluorescent emission. As more PCR product is created, the intensity of the novel wavelength increases. While TaqMan accomplishes the goal of single nucleotide detection in a homogenous assay, it has two disadvantages. The first is that each nucleotide to be detected requires a different oligonucleotide probe comprising two different fluorescent moieties. Such probes must be custom-synthesized and are thus expensive. The second disadvantage is that TaqMan probes are not very discriminating for single nucleotide differences. Thus there can be significant false-positive signals.
Molecular Beacons are an alternative to TAQMAN (U.S. Pat. Nos. 6,277,607; 6,150,097; 6,037,130). Molecular Beacons undergo a conformational change upon binding to a perfectly matched template. The conformational change of the Beacon increases the physical distance between a fluorophore moiety and a quencher moiety on the Beacon. This increase in physical distance causes the effect of the quencher to be diminished, thus increasing the signal derived from the fluorophore. Molecular Beacons are more discriminating of single nucleotide differences, as compared with TaqMan probes. However they still require the synthesis of a custom oligonucleotide (the Beacon) having two different fluorescent moieties for each target sequence being examined. Thus the technology is expensive.
There are several other fluorescent and enzymatic PCR technologies, such as SCORPIONS(trademark), SUNRISE(trademark) primers, and DNAzymes. Not all of these are suitable for single nucleotide detection, and most of them require the synthesis of a custom, fluorescently labeled oligonucleotide for each target nucleotide.
Hybridization to a xe2x80x9cDNA chipxe2x80x9d is another way of detecting single nucleotide differences (U.S. Pat. No. 5,856,104). Typically oligonucleotides that are complementary to the suspected target DNAs are synthesized on a solid surface (xe2x80x9cchipxe2x80x9d or xe2x80x9coligonucleotide arrayxe2x80x9d). The target DNA is PCR amplified, labeled, and then hybridized to the oligonucleotide array. Ideally, perfectly matched PCR fragments will hybridize to the array, but mismatched fragments will not. While the technology, in theory, offers the opportunity to look at many different loci simultaneously, in practice the need to amplify the target DNA using PCR limits the degree to which the assay can be multiplexed. In addition the start-up costs for designing an oligonucleotide microarray can be very expensive. Lastly, the frequency of false-positive and false-negative spots is very high, and necessitates the use of many surface-bound oligonucleotides for each target DNA sequence.
There currently are two non-PCR based technologies capable of detecting single nucleotide changes in complex genomes. The Invader-Squared method (U.S. Pat. No. 6,001,567) utilizes a cascade of DNA cleavage reactions. While sensitive, it requires the synthesis of several long, target-specific oligonucleotides in addition to several detection oligonucleotides. The rolling circle detection method (Lizardi et al., Nature Genetics 19: 225-232) utilizes a target nucleotide-specific ligation reaction to create a circular template that is then replicated with a polymerase in rolling-circle fashion. One of the advantages is that the reaction does not require thermal cycling. One drawback is that ligation reactions are not highly specific for single nucleotide detection.
Single base extension (xe2x80x9cSBExe2x80x9d; also called minisequencing) is a technology that uses dideoxy chain terminators in combination with a DNA polymerase to determine the identity of a single nucleotide in a target DNA sample that has been PCR amplified (Syvanen et al., 1990, Genetics 8:684-642; U.S. Pat. No. 5,888,819; Euoropean patent application EP 0648280 A1, each of which is incorporated herein by reference). The technology uses a DNA primer that is hybridized to a target polynucleotide in the presence of dideoxy chain terminators, but typically in the absence of deoxynucleotide triphosphates. A DNA polymerase will add a single dideoxy chain terminator to the 3xe2x80x2 end of a primer that is reasonably hybridized to the DNA target. The polymerase incorporates the appropriate dideoxy terminator determined by the complementary sequence in the target polynucleotide. Thus, the identity of the dideoxy terminator that is incorporated reflects the identity of the nucleotide within the target polynucleotide that is immediately adjacent to the target nucleotide that is hybridized with the 3xe2x80x2 nucleotide of the primer.
There are a number of patents and patent applications for SBE. In U.S. Pat. No. 6,013,431, the dideoxy chain terminators would be labeled with reporter moieties, such as fluorescent molecules, and the incorporation of a label into a primer is measured by gel electrophoresis. The method described in U.S. Pat. Nos. 6,015,675; 5,582,989; 5,578,458 relates to placing the primer on a solid surface, such as a chip. The chip is exposed to a solution containing the target polynucleotide plus fluorescently labeled dideoxy chain terminators and polymerase. When a single labeled base is added to the bound primer, the probe begins to fluoresce.
Fluorescence polarization has been used to perform SBE. With this approach the chain terminators are fluorescently labeled as with other methods. However rather than separating the labeled primers by gel electrophoresis or physical separation, the incorporated chain terminators are generated by shining polarized light on the sample, and then detecting the polarization of the emitted fluorescent light. Fluorescent light emitted by unincorporated terminators will not be polarized because these small molecules are rapidly moving in solution. However labeled terminators that have been incorporated onto the end of a primer will be moving more slowly and tend to emit polarized light. Thus the degree to which the emitted light is polarized reflects the degree to which there has been incorporation of a dideoxy chain terminator onto the end of a primer. The color of the polarized emitted light reflects the particular dideoxy terminator (A, C, G, or T) that was incorporated onto the 3xe2x80x2 end of the primer. The advantage to the fluorescent polarization method is that it is homogeneous (all done in a single test tube). However the input target DNA is typically a PCR fragment, and the PCR reaction needs to be performed prior to SBE. Moreover the PCR product needs to be separated from the PCR primers and deoxynucleotides of the PCR reaction prior to performing the SBE reaction.
Another homogenous method has been described in U.S. Pat. No. 6,177,249. This patent uses fluorescence resonance energy transfer (xe2x80x9cFRETxe2x80x9d) (Wittwer, et al., 1997, Biotechniques 22:130-138; Bernard, et al., 1998, Am. J. Pathol. 153:1055-1061). FRET occurs when two fluorescent molecules are in close physical proximity (e.g., 10-100 xc3x85), and one of the fluorescent molecules can absorb light of a wavelength that is emitted by the other fluorescent molecule. For example, suppose the first fluorescent molecular is stimulated by blue light and emits green light, and the second fluorescent molecule is stimulated by green light and emits red light. If, for example, an oligonucleotide contains both fluorescent molecules and the primer is illuminated with blue light, it will emit red light without emitting much green light. In U.S. Pat. No. 6,177,249 (supra), the SBE primer contains one fluorescent molecule. The dideoxy chain terminators contain another (up to 4 different) fluorescent molecules. Upon addition of a terminator to the 3xe2x80x2 end of a primer, FRET can occur. As per the FRET example above, stimulating blue light would be converted to green light by the fluorophore on the primer, and then would be further converted to red light after a terminator has been added to the primer. The emission of red light would be used to monitor the degree to which terminators have been added to the primer. One would use 4 terminators with 4 different emission spectra, but all capable of being stimulated by the wavelength released by the primer-bound fluorophore. The advantage to this method is that it is a homogenous assay, although still requiring a PCR amplification pre-SBE step for complex genomes. The disadvantage is that the user must synthesize an expensive, custom oligonucleotide primer for each target DNA locus being examined.
The present invention relates to compositions and methods for the detection of nucleotides at predetermined locations on a polynucleotide of interest. The embodiments of the invention include compositions and methods in which a primer extension reaction is designed to extend a single nucleotide (single base extension, SBE) and the incorporation of a labeled chain terminator is determined by signal transfer.
The invention provides a composition for identifying a nucleotide at a predetermined position of a target polynucleotide in a sample, the composition comprising:
(a) an oligonucleotide primer comprising a first sequence which hybridizes to the target polynucleotide immediately 3xe2x80x2 of the nucleotide, and a second sequence which does not hybridize to the target polynucleotide in the presence of a third sequence; and
(b) an oligonucleotide probe comprising the third sequence which hybridizes to the second sequence of the oligonucleotide primer, the oligonucleotide probe labeled with a first member of a pair of interactive labels.
The second sequence of the oligonucleotide primer is preferably located at the 5xe2x80x2 terminal of the first sequence.
The composition of the invention may also comprise a first polynucleotide chain terminator, which is incorporated in a template-dependent manner into the oligonucleotide primer by a polynucleotide synthesis enzyme.
The composition of the invention may further comprise one or more of a second, a third and/or a fourth polynucleotide chain terminator, where the first, second, third and fourth polynucleotide terminators are not identical.
The composition of the invention may still further comprises a template-dependent polynucleotide synthesis enzyme for incorporating in a template-dependent manner a complementary polynucleotide chain terminator into the oligonucleotide primer.
Preferably, the first polynucleotide chain terminator of the subject composition is labeled with a second member of the pair of interactive labels.
In a preferred embodiment, one member of the pair of interactive labels is a quencher molecule.
In one embodiment of the invention, the first and second members of the pair of interactive labels interact with each other to generate a signal by fluorescent resonance energy transfer.
Preferably, the first and second members of the pair of interactive labels are fluorescent molecules which interact with each other to generate a signal by fluorescent resonance energy transfer.
Also preferably, the polynucleotide synthesis enzyme of the subject composition is a JDF-3 DNA polymerase.
In one embodiment of the invention, the oligonucleotide primer comprises a separation moiety that permits separation of the oligonucleotide primer and/or the oligonucleotide probe hybridized to the primer from unincorporated polynucleotide chain terminator, and oligonucleotide probe which is not hybridized to the oligonucleotide primer.
Preferably, the composition of the subject invention also provides a target moiety specific for the separation moiety, where the separation moiety binds to the target moiety to permit the separation.
The target moiety of the composition is preferably attached to a solid support.
The invention provides another composition for identifying a nucleotide at a predetermined position of a target polynucleotide in a sample, the composition comprising:
(a) an oligonucleotide primer comprising a first sequence which hybridizes to the target polynucleotide immediately 3xe2x80x2 of the nucleotide, and is covalently attached to a tag molecule; and
(b) an anti-tag molecule which binds to the tag molecule, the anti-tag molecule labeled with a first member of a pair of interactive labels.
The tag molecule of the subject composition is preferably located on the 5xe2x80x2 terminal of the oligonucleotide primer.
Preferably, the tag molecule is a first member of a specific binding pair which comprises the first member and a second member.
Also preferably, the anti-tag molecule is the second member of the specific binding pair.
In one embodiment, the specific binding pair is a biotin-streptavidin pair.
The invention provides a kit for identifying a nucleotide at a predetermined position of a target polynucleotide in a sample, the kit comprising:
(a) an oligonucleotide primer comprising a first sequence which hybridizes to the target polynucleotide immediately 3xe2x80x2 of the nucleotide, and a second sequence which does not hybridize to the target polynucleotide in the presence of a third sequence;
(b) an oligonucleotide probe comprising the third sequence which hybridizes to the second sequence of the oligonucleotide primer, the oligonucleotide probe labeled with a first member of a pair of interactive labels; and
(c) packaging materials therefore.
The kit of the subject invention may also comprise a polynucleotide chain terminator, which can be incorporated in a template-dependent manner into the oligonucleotide primer by a polynucleotide synthesis enzyme.
The kit of the subject invention may further comprise one or more of a second, a third and/or a fourth polynucleotide chain terminator, where the first, second, third and fourth polynucleotide terminators are not identical.
The polynucleotide chain terminator of the kit is preferably labeled with a second member of the pair of interactive labels.
The kit of the subject kit may still further comprise a template-dependent polynucleotide synthesis enzyme for incorporating in a template-dependent manner a complementary polynucleotide chain terminator into the oligonucleotide primer.
Preferably, the polynucleotide synthesis enzyme is a JDF-3 DNA polymerase.
The invention provides a kit for identifying a nucleotide at a predetermined position of a target polynucleotide in a sample, the kit comprising:
(a) an oligonucleotide primer comprising a first sequence which hybridizes to the target polynucleotide immediately 3xe2x80x2 of the nucleotide, and is covalently attached to a tag molecule;
(b) an anti-tag molecule which binds to the tag molecule, the anti-tag molecule being labeled with a first member of a pair of interactive labels; and
(c) packaging materials therefore.
The tag molecule of the subject kit is preferably a first member of a specific binding pair which comprises the first member and a second member.
Preferably, the anti-tag molecule is the second member of the specific binding pair.
In one embodiment of the invention, the specific binding pair comprises a biotin-streptavidin pair.
The invention provides a method of identifying the presence of a nucleotide at a predetermined position of a target polynucleotide, the method comprising:
(a) incubating the target polynucleotide in a reaction mixture comprising an oligonucleotide primer which hybridizes to the target polynucleotide immediately 3xe2x80x2 of the nucleotide, an oligonucleotide probe which hybridizes to the oligonucleotide primer and labeled with a first member of a pair of interactive labels, a polynucleotide chain terminator labeled with a second member of the pair of interactive labels, where the incubating permits the polynucleotide chain terminator to be incorporated into the oligonucleotide primer, and permits the oligonucleotide probe to hybridize to the oligonucleotide primer to permit the pair of interactive labels to generate a signal; and
(b) detecting the signal, where the detection is indicative of the presence of the nucleotide in the target polynucleotide.
The invention also provides a method of identifying the presence of a nucleotide at a predetermined position of a target polynucleotide, the method comprising the steps:
(a) incubating the target polynucleotide in a reaction mixture comprising an oligonucleotide primer which hybridizes to the target polynucleotide immediately 3xe2x80x2 of the nucleotide and a polynucleotide chain terminator labeled with a second member of a pair of interactive labels, where the incubating permits the polynucleotide chain terminator to be incorporated into the oligonucleotide primer;
(b) incubating the oligonucleotide primer comprising the second member of the pair of interactive labels with an oligonucleotide probe labeled with a first member of the pair of interactive labels, such that formation of a hybrid between the oligonucleotide probe and the primer permits the pair of interactive labels to a generate a signal; and
(c) detecting the signal, where the detection is indicative of the presence of the nucleotide in the target polynucleotide.
In one embodiment of the invention, the signal is generated by fluorescent resonance energy transfer.
In a preferred embodiment, the oligonucleotide primer comprises a first sequence which hybridizes to the target polynucleotide and a second sequence which does not hybridize to the target polynucleotide in the presence of a third sequence.
Preferably, the second sequence on the oligonucleotide primer is located at the 5xe2x80x2 terminal of the first sequence.
Also preferably, the oligonucleotide probe comprises the third sequence which hybridizes to the second sequence of the oligonucleotide primer.
In one embodiment, the polynucleotide chain terminator is incorporated by a polynucleotide synthesis enzyme.
The reaction mixture of the subject method may also comprise one or more of a second, a third and/or a fourth polynucleotide chain terminator, where the first, second, third and fourth polynucleotide terminators are not identical.
Preferably, the polynucleotide synthesis enzyme is a JDF-3 DNA polymerase.
The oligonucleotide primer of the subject method may comprise a separation moiety that permits separation of the oligonucleotide primer from the reaction mixture.
Preferably, a target moiety is provided in the subject method for the separation moiety to form a specific binding pair for separation.
In one embodiment, the target moiety is attached to a solid support.
The invention provides a method for identifying the presence of a nucleotide at a predetermined position of a target polynucleotide, the method comprising:
(a) incubating the target polynucleotide in a reaction mixture comprising an anti-tag molecule labeled with a first member of a pair of interactive labels, a polynucleotide chain terminator labeled with a second member of the pair of interactive labels, and an oligonucleotide primer which hybridizes to the target polynucleotide immediately 3xe2x80x2 of the nucleotide, the oligonucleotide primer covalently coupled to a tag molecule, where the incubating permits the polynucleotide chain terminator to be incorporated into the oligonucleotide primer, and the incubating also permits the anti-tag molecule to interact with the tag molecule on the oligonucleotide primer, so that the pair of interactive labels generate a signal; and
(b) detecting the signal, where the detection is indicative of the presence of the nucleotide in the target polynucleotide.
In a preferred embodiment, the signal is generated by fluorescent resonance energy transfer.
In another preferred embodiment, one member of the pair of interactive labels is a quencher molecule.
Preferably, the tag molecule is located at 5xe2x80x2 terminal of the oligonucleotide primer.
The tag molecule of the subject method may comprise a first member of a specific binding pair which comprises the first member and a second member.
The anti-tag molecule may comprise the second member of the specific binding pair.
In one embodiment, the specific binding pair is a biotin-streptavidin binding pair.
The chain terminator of the invention may be one selected from the group consisting of: a dideoxynucleotide triphosphate, a ribofuranose analog, a reversible nucleotide terminator, and an acyclic terminator.
The target polynucleotide of the invention may present in a sample.